A Novel Immune Evasion Strategy of Candida albicans: Proteolytic Cleavage of a Salivary Antimicrobial Peptide

Oropharyngeal candidiasis is an opportunistic infection considered to be a harbinger of AIDS. The etiologic agent Candida albicans is a fungal species commonly colonizing human mucosal surfaces. However, under conditions of immune dysfunction, colonizing C. albicans can become an opportunistic pathogen causing superficial or even life-threatening infections. The reasons behind this transition, however, are not clear. In the oral cavity, salivary antimicrobial peptides are considered to be an important part of the host innate defense system in the prevention of microbial colonization. Histatin-5 specifically has exhibited potent activity against C. albicans. Our previous studies have shown histatin-5 levels to be significantly reduced in the saliva of HIV+ individuals, indicating an important role for histatin-5 in keeping C. albicans in its commensal stage. The versatility in the pathogenic potential of C. albicans is the result of its ability to adapt through the regulation of virulence determinants, most notably of which are proteolytic enzymes (Saps), involved in tissue degradation. In this study, we show that C. albicans cells efficiently and rapidly degrade histatin-5, resulting in loss of its anti-candidal potency. In addition, we demonstrate that this cellular activity is due to proteolysis by a member of the secreted aspartic proteases (Sap) family involved in C. albicans pathogenesis. Specifically, the proteolysis was attributed to Sap9, in turn identifying histatin-5 as the first host-specific substrate for that isoenzyme. These findings demonstrate for the first time the ability of a specific C. albicans enzyme to degrade and deactivate a host antimicrobial peptide involved in the protection of the oral mucosa against C. albicans, thereby providing new insights into the factors directing the transition of C. albicans from commensal to pathogen, with important clinical implications for alternative therapy. This report characterizes the first defined mechanism behind the enhanced susceptibility of HIV+ individuals to oral candidiasis since the emergence of HIV

Candida albicans Scavenges Host Zinc via Pra1 during Endothelial Invasion

The ability of pathogenic microorganisms to assimilate essential nutrients from their hosts is critical for pathogenesis. Here we report endothelial zinc sequestration by the major human fungal pathogen, Candida albicans. We hypothesised that, analogous to siderophore-mediated iron acquisition, C. albicans utilises an extracellular zinc scavenger for acquiring this essential metal. We postulated that such a “zincophore” system would consist of a secreted factor with zinc-binding properties, which can specifically reassociate with the fungal cell surface. In silico analysis of the C. albicans secretome for proteins with zinc binding motifs identified the pH-regulated antigen 1 (Pra1). Three-dimensional modelling of Pra1 indicated the presence of at least two zinc coordination sites. Indeed, recombinantly expressed Pra1 exhibited zinc binding properties in vitro. Deletion of PRA1 in C. albicans prevented fungal sequestration and utilisation of host zinc, and specifically blocked host cell damage in the absence of exogenous zinc. Phylogenetic analysis revealed that PRA1 arose in an ancient fungal lineage and developed synteny with ZRT1 (encoding a zinc transporter) before divergence of the Ascomycota and Basidiomycota. Structural modelling indicated physical interaction between Pra1 and Zrt1 and we confirmed this experimentally by demonstrating that Zrt1 was essential for binding of soluble Pra1 to the cell surface of C. albicans. Therefore, we have identified a novel metal acquisition system consisting of a secreted zinc scavenger (“zincophore”), which reassociates with the fungal cell. Furthermore, functional similarities with phylogenetically unrelated prokaryotic systems indicate that syntenic zinc acquisition loci have been independently selected during evolution

The Glycosylphosphatidylinositol-Anchored Protease Sap9 Modulates the Interaction of Candida albicans with Human Neutrophils ▿

Human polymorphonuclear neutrophils (PMNs) play a major role in the immune defense against invasive Candida albicans infection. This fungal pathogen produces a set of aspartic proteases that directly contributes to virulence properties such as adhesion, tissue invasion, and immune evasion. We show here that, in contrast to other secreted proteases, the cell surface-associated isoform Sap9 has a major impact on the recognition of C. albicans by PMNs. SAP9 is required for the induction of PMN chemotaxis toward C. albicans filaments, an essential prerequisite of effective PMN activation. Furthermore, deletion of SAP9 leads to a mitigated release of reactive oxygen intermediates (ROI) in human PMNs and decreases C. albicans-induced apoptosis triggered by ROI formation. In confrontation assays, killing of a SAP9 deletion mutant is reduced in comparison to wild-type C. albicans. These data clearly implicate Sap9 protease activity in the initiation of protective innate immunity and suggest novel molecular mechanisms in C. albicans-host interaction leading to neutrophil activation

The Inflammatory Response Induced by Aspartic Proteases of Candida albicans Is Independent of Proteolytic Activity ▿

The secretion of aspartic proteases (Saps) has long been recognized as a virulence-associated trait of the pathogenic yeast Candida albicans. In this study, we report that different recombinant Saps, including Sap1, Sap2, Sap3, and Sap6, have differing abilities to induce secretion of proinflammatory cytokines by human monocytes. In particular Sap1, Sap2, and Sap6 significantly induced interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6 production. Sap3 was able to stimulate the secretion of IL-1β and TNF-α. All Saps tested were able to induce Ca2+ influx in monocytes. Treatment of these Saps with pepstatin A did not have any effect on cytokine secretion, indicating that their stimulatory potential was independent from their proteolytic activity. The capacity of Saps to induce inflammatory cytokine production was also independent from protease-activated receptor (PAR) activation and from the optimal pH for individual Sap activity. The interaction of Saps with monocytes induced Akt activation and phosphorylation of IκBα, which mediates translocation of NF-κB into the nucleus. Overall, these results suggest that individual Sap proteins can induce an inflammatory response and that this phenomenon is independent from the pH of a specific host niche and from Sap enzymatic activity. The inflammatory response is partially dependent on Sap denaturation and is triggered by the Akt/NF-κB activation pathway. Our data suggest a novel, activity-independent aspect of Saps during interactions of C. albicans with the host

Phase 1 Study of Accelerated Hypofractionated Radiation Therapy With Concurrent Chemotherapy for Stage III Non-Small Cell Lung Cancer: CALGB 31102 (Alliance).

PurposeTo investigate the safety of accelerated hypofractionated radiation therapy (AHRT) with concurrent chemotherapy (CT) for inoperable stage III non-small cell lung cancer (NSCLC).Patients and methodsThe primary objectives were to define the maximally tolerable course of accelerated radiation therapy and to describe toxicities of therapy. Total radiation therapy remained at 60 Gy. The number of once-daily fractions in each successive cohort was reduced as follows: cohort 1, 60 Gy in 27 fractions; cohort 2, 60 Gy in 24 fractions; cohort 3, 60 Gy in 22 fractions; and cohort 4, 60 Gy in 20 fractions. Concurrent treatment consisted of weekly carboplatin area under the curve (AUC) 2 and paclitaxel 45 mg/m2. Consolidation treatment consisted of carboplatin AUC 6 and paclitaxel 200 mg/m2 every weeks × 2 cycles. Maximum tolerated dose: Of 6 patients/cohort, ≤2 patients experienced grade ≥3 toxicity, and ≤1 patient experienced grade ≥4 toxicity.Results22 patients were accrued; of those, 21 patients were evaluable between July 2012 and May 2014. Grade 5 toxicity occurred in 3 patients: 1 patient in cohort 2 (hemoptysis), 2 patients in cohort 3 (hemoptysis, pneumonitis). The maximum tolerated dose (MTD) was defined by cohort 2 (60 Gy in 2.5 Gy/fraction). Time to grade 5 toxicity was 9 months, 6 months, and 9 months after the start of treatment. The median follow-up time was 23.0 months (range, 7.6-30.6 months) in living patients, the median overall survival was 19.3 months (95% confidence interval [CI] 9.3-34.0 months), and the median progression-free survival was 12.2 months (95% CI 6.1-22.5 months).ConclusionsOnly modest hypofractionation was achievable as a result of long-term toxicities. Nevertheless, the MTD of 60 Gy given at 2.5 Gy/fraction allows completion of RT in 20% fewer treatments than conventional therapy. Further investigation of AHRT may help to better define the therapeutic index

<i>In silico</i> prediction of Pra1-zinc binding.

<p>(<b>A</b>) Primary amino acid sequence of Pra1 (<i>Candida</i> Genome Database) with zinc-binding motifs in red. (<b>B</b>) Three-dimensional model of Pra1 built with Phyre². (<b>C</b>) and (<b>D</b>) close-up of predicted zinc coordination sites. Note the presence of arginine, rather than glutamic acid at residue 179 (<b>C</b>) and that the C-terminal tail of Pra1 has multiple additional potential zinc binding histidine residues (<b>D</b>).</p

<i>ZRT1</i> and <i>PRA1</i> are required for microcolony development on endothelia in the absence of exogenous zinc.

<p>Single cells of <i>C. albicans</i> wild type (M1477), <i>zrt1</i>Δ (M2006) or <i>pra1</i>Δ (M2008) were incubated for 16 h in zinc-depleted cell culture medium on endothelial monolayers (Endothelium) or on plastic (Ctrl). Experiment was performed three times. representative images are shown. Note that only wild type cells were able to assimilate sufficient zinc for microcolony development.</p

Model of <i>C. albicans</i> zinc scavenging from host cells.

<p>Following host cell invasion, Pra1 is expressed and secreted. The released fraction of Pra1 binds zinc, either directly from a cellular pool or from host zinc-binding proteins. Reassociation with the cell surface of <i>C. albicans</i> is mediated via direct Pra1-Zrt1 interaction.</p

Endothelial damage is zinc-dependent in the absence of <i>PRA1</i>.

<p>Endothelial monolayers were infected with wild type (M134), <i>pra1</i>Δ (M1809) or <i>pra1</i>Δ+<i>PRA1</i> (M1785) <i>C. albicans</i> in zinc-free medium with indicated zinc-supplementation. Following 24 h co-incubation, endothelial damage was assessed by measuring the release of lactate dehydrogenase. Experiment was performed 3 times in triplicate. Asterisks indicate significance by Student's t-test: * <0.05; ** <0.01; *** <0.001.</p